Biomedical-engineering advancements have provided a variety of tools to explore the detailed structure and behavior of biological tissues. Traditional equipment in this area has provided images and other data by way of x-rays, sound waves, and visible and infrared (IR) light to characterize the structure and behavior of certain tissues. Although generally successful, the image quality provided by such conventional equipment is limited and not applicable to ail types of biological tissues. As examples, X-ray equipment typically transmits relatively low-level radiation and is used to characterize the location of the tissue as a function of its periphery, and visible/IR light imaging tools are used for characterizing transparent and semi-transparent tissue but are ineffective for imaging optically-dense (“thick”) tissue.
Conventional approaches for high-resolution images of thick tissue have not been widely implemented due to approach-specific issues. Generally, these approaches can be categorized as “transmission-mode” (a.k.a., “forward-direction”) systems and “reverse-direction” systems. Transmission-mode systems radiate energy at the tissue from one side and use a nearby sensing device on the opposite side of the tissue to sense the radiated energy after it is impacted by the tissue. One form of forward-direction imaging relies on SHG (second harmonic generation) which is known to be a forward-directed nonlinear optical process. In SHG, a light source directs photons at a target material for interacting and combining into higher-energy photons. The higher-energy photons are predominantly forwardly-directed at a sensing device on the opposite side of the tissue. While useful for many in vitro applications, this transmission-mode approach can be extremely invasive due to the need for a sensing device on the opposite side of the tissue. In more tissue-sensitive applications such as in vivo examinations and in vitro investigations where the integrity of the tissue is to be maintained after examination, this approach would be unacceptable due to the placement of the sensing device deep within the subject under examination.
Reverse-direction systems radiate energy at the tissue from one side and use a sensing device on the same side of the tissue to sense energy radiated in response. Unlike transmission-mode systems, these systems do not require placement of a sensing device on the opposite side of the tissue and therefore could be considered less invasive for in vivo applications. For high-resolution imaging of thick tissue, however, these systems require relatively strong signals and can require pre-treatment of the tissue with a foreign matter (e.g., dye, exogenous gene or protein) in order to enhance signals responding to excitation of the tissue by light. Such pre-treatment is undesirable for reasons concerning the invasiveness of the foreign matter and its alteration of the cells under examination.
Recent attempts to use reverse-direction systems have not been widely adopted. These attempts have relied on back-directed SHG or on endogenous (or native) fluorescence for tissue characterization for a variety of reasons. These approaches are burdened by insufficient signal strengths and/or the need to physically mitigate physiological motions associated with blood flow and respiratory activity. For imaging skeletal and/or cardiac muscle tissues, motions associated with sarcomere contractions further perturb image quality.